Many human diseases and conditions are caused by gene mutations. Substantial effort has been directed towards the creation of transgenic animal models of such diseases and conditions to facilitate the testing of approaches to treatment, as well as to gain a better understanding of disease pathology. Early transgenic animal technology focused on the mouse, while more recent efforts, which have been bolstered by the development of somatic cell nuclear transfer (SCNT), have included larger animals, including pigs, cows, and goats. This technology has resulted in the production of, for example, pigs in which the gene encoding α-1,3-galactosyltransferase has been knocked out, in efforts to generate organs that can be used in xenotransplantation (see, e.g., Lai et al., Science 295:1089-1092, 2002). Further, this technology has resulted in the production of large animal models of human cystic fibrosis (CFTR−/− and CFTR-ΔF508/ΔF508 pigs, see, e.g., U.S. Pat. Nos. 7,989,675 and 8,618,352, and U.S. patent application Ser. Nos. 13/368,312 and 13/624,967); and large animal models of human cardiovascular disease (LDLR+/+ and LDLR −/− pigs, see, e.g., U.S. patent application Ser. No. 13/368,312). Additional applications of this technology include the production of large quantities of human proteins (e.g., therapeutic antibodies; see, e.g., Grosse-Hovest et al., Proc. Natl. Acad. Sci. U.S.A. 101(18): 6858-6863, 2004). Substantial benefits may be obtained by the use of somatic cell nuclear transfer technology in the production of large animal models of human disease.
Cancer is the second most common cause of death in the United States, killing over 500,000 children and adults each year. See, e.g., Jemal, A., et al., CA Cancer J Clin (2010). Nearly 11 million Americans have a history of cancer, and an additional 1.5 million cases are diagnosed annually. The NIH estimates the annual overall cost of cancer to be more than $200 billion. The National Cancer Institute spends $4.8 billion annually on cancer research with additional funds coming from private industry and disease foundations. See, e.g., National Cancer Institute. Cancer Research Funding. 2010 (www.cancer.gov/cancertopics/factsheet/NCI/research-funding#a5). Yet, despite these significant expenditures, current treatments remain inadequate. All too often, therapeutic strategies that show promise in the current preclinical model systems fail to yield results in patients. This is particularly true with cancer where less than 5% of treatments that enter clinical trials are approved for use in humans. Kola, I., et al., Nat Rev Drug Discov 2004, 3 (8), 711-5; Hackam, D. G., et al., JAMA 2006, 296 (14), 1731-2. This lack of predictive efficacy in the drug development process is costly, with over 70% of all drug development costs being the result of failed drugs. An animal model that accurately replicates the progression of human cancer and shares similarities to humans in size, anatomy, physiology, and genetics would bridge the substantial gap between models currently used for early-stage drug discovery and Phase 0/I human clinical trials.
Beyond the current inefficiency in the treatment development cycle, significant challenges also remain in determining which clinically approved treatment strategy is best for each patient and confirming whether the chosen approach is effective. Non-invasive medical imaging methods have great potential to facilitate cancer treatment through lesion detection, characterization, treatment planning and monitoring. However, development and validation of new medical imaging technologies are also limited by the current model systems. Rodent models are not well suited for these applications due to their size, and current large animal models are insufficient because cancer must be induced with long-term, high-dose chemical carcinogens or tissue grafting procedures that do not resemble naturally occurring human tumors.
It has been observed in mice that carcinogenesis depends on the activation of proto-oncogenes and the deactivation of tumor suppressor genes. A mutation leading to the activation of an oncogene alone (for example, the KRAS gene) will not necessarily lead to the development of cancer, as normally functioning tumor suppressor genes (TSGs) would still function to maintain normal cell cycle. However, if TSGs are also damaged, leading to the inactivation of certain tumor suppressor proteins, unchecked cell proliferation results and leads to cancer. Conversely, a damaged TSG (for example, TP53 and ATM) would not necessarily result in cancer decoupled from the uncontrolled growth resulting from an activated oncogene.
While these observations in murine models have been helpful, they do not provide an adequate model for the development and progression of cancer in humans. Thus, a large animal model (for example, a porcine model) that is predisposed to numerous types of cancer would benefit multiple disciplines within the cancer research community. In one example, because p53 dysfunction is associated with more than half of all cancers, a large animal model with a mutation in the TSG TP53 will serve as a platform for the development of many specific cancer models. See, e.g., Bartek, J., et al., Oncogene 1991, 6 (9), 1699-703.
In another example, mutations in the Ataxia-Telangiectasia Mutated (ATM) gene are also associated with the development of certain types of cancer. It is known, for example, that individuals with ataxia-telangiectasia (A-T) (ATM −/−) are estimated to have a 100-fold increased risk of cancer compared with the general population. Lymphoid cancers predominate in childhood, and epithelial cancers, including breast cancer, are seen in adults. See Ahmed, M., et al., Onconogene (2006) 25, 5906-5911. It is also known that people who have only one copy of the ATM gene in each cell (ATM +/−) are at an increased risk of developing breast cancer and may have an increased risk of developing other types of cancer, for example, stomach, bladder, pancreas, lung, and ovarian. See, e.g., Shen L., et al., Mol Biol Rep. 2012 May; 39(5):5719-25. Cells that are missing one copy of the ATM gene produce half the normal amount of ATM protein, which prevents efficient repair of DNA damage and leads to the accumulation of mutations in other genes. Thus, a large animal model with a mutation in ATM would serve as a platform for the development of many specific cancer models.
In yet another example, large animal models in which tumorigenesis can be initiated in a tissue-specific manner will benefit multiple disciplines within the cancer research community. It is known, for example, that mutations in the KRAS oncogene are associated with about 30% of all human cancers Thus, an animal model having one or more mutations in the KRAS gene can be used to develop a large animal model in which tumorigenesis can be initiated in a tissue-specific manner.
In further examples of a large animal model of cancer, specific mutations introduced into a cancer-prone animal model (for example, a mutated TP53 or ATM animal model) would be extremely useful for the development of specific cancer models. In this example, a mutation in one or more genes associated with the development of cancer (for example, KRAS, TP53 and ATM) can be introduced in a large animal model having a mutation in a different gene associated with cancer development (for example, KRAS, TP53 or ATM).
Thus, in one example, because mutations in TP53 and KRAS are associated with more than half of all cancers, a large animal model with targeted mutations in the TP53 and KRAS genes would serve as a platform for the development of many specific cancer models. Bartek, J., et al., Oncogene 1991, 6 (9), 1699-703. Malumbres, M., et al., Nat Rev Cancer 2003, 3 (6), 459-65. In a similar example, a large animal model having one or more mutations in TP53 and ATM may also serve as a platform for the development of certain cancer models.
TP53 Animal Model Platform
p53 (protein 53 or tumor protein 53) is strongly associated with the development of cancer in humans. Functional p53 regulates the cell division cycle and serves as a tumor suppressor in cells. p53 is activated in cells by certain stress events, for example, DNA damage, oxidative stress, osmotic shock, ribonucleotide depletion, and deregulated oncogene expression. If the TP53 gene is damaged, tumor suppression is severely reduced. For example, more than 50 percent of tumors are associated with a mutation or deletion of the TP53 gene. See Hollstein, M., et al., Science. 1991; 253(5015):49-53. Further, those who inherit only one functional copy of the TP53 gene will most likely develop tumors in early adulthood, a disease known as Li-Fraumeni syndrome.
Unlike the majority of tumor suppressor genes, such as RB, APC, or BRCA1, which are usually inactivated during cancer progression by deletions or truncating mutations, the TP53 gene in human tumors is often found to undergo missense mutations, in which a single nucleotide is substituted by another. Consequently, a full-length protein containing only a single amino acid substitution is produced. The cancer-associated TP53 mutations are very diverse in their locations within the p53 coding sequence and their effects on the thermodynamic stability of the p53 protein. However, the vast majority of the mutations result in loss of p53's ability to bind DNA in a sequence-specific manner and activate transcription of canonical p53 target genes. See Hainaut P, et al., Adv Cancer Res. 2000; 77:81-137; Bullock A. N., et al., Nat Rev Cancer. 2001; 1:68-76; Rivlin, N., et al., Genes & Cancer 2011 2: 466.
TP53 mutations are distributed in all coding exons of the TP53 gene, with a strong predominance in exons 4-9, which encode the DNA-binding domain of the protein. Of the mutations in this domain, about 30% fall within 6 “hotspot” residues (residues R175, G245, R248, R249, R273, and R282) and are frequent in almost all types of cancer. See Cho Y., et al., Science. 1994; 265:346-55. The existence of these hotspot residues could be explained both by the susceptibility of particular codons to carcinogen-induced alterations and by positive selection of mutations that render the cell with growth and survival advantages.
It is well established that p53 inactivation and mutant p53 expression can grant cells with additive growth and survival advantages, such as increased proliferation, evasion of apoptosis, and chemoresistance. See Sigal A., et al., Cancer Res. 2000; 60:6788-93; Brosh R., et al., Nat Rev Cancer. 2009; 9:701-13. In an effort to further study the mechanisms that underlie the role of mutant p53 at the various steps of tumor progression, it is important to establish animal models that express mutant p53 in a controlled manner. Recent data obtained through the use of such in vivo models support the notion of gain of function properties acquired by mutant p53, which drive cells toward migration, invasion, and metastasis.
Earlier work revealed that although p53 knockout mice develop tumors at a high frequency, they exhibit a rather low occurrence of metastasis or invasive growth. See Donehower L. A., et al., Nature. 1992; 356:215-21; Attardi L. D., et al., Cell Mol Life Sci. 1999; 55:48-63. In contrast to this, mice knocked in with p53 R270H or R172H, corresponding to the human hotspot mutants p53R273H and p53R175H, respectively, developed highly metastatic tumors. See Lang G. A., et al., Cell. 2004; 119:861-72; Heinlein C, et al., Int J Cancer. 2008; 122:1701-9. These data support the hypothesis that TP53 mutations at early stages of tumorigenesis contribute mainly to uncontrolled proliferation, a feature of both benign and malignant tumors, whereas mutations at later stages synergize with additional oncogenic events to drive invasion and metastasis, the hallmark of malignant tumors.
While murine models of p53 mutations have provided valuable insights into the development and progression of cancer, a large animal model that more closely resembles human biology, for example, metabolism, physiology, and tumor biology, is needed to advance our understanding of cancer development and progression.
KRAS Tissue Specific Animal Model
KRAS is a potent oncogene and is mutated in about 30% of all human cancers. However, the biological context of KRAS-dependent oncogenesis is poorly understood. Genetically engineered mouse models of cancer have provided some tools to study the oncogenic process, and insights from KRAS-driven models have significantly increased understanding of the genetic, cellular, and tissue contexts in which KRAS is competent for oncogenesis. Moreover, variation among tumors arising in mouse models can provide insight into the mechanisms underlying response or resistance to therapy in KRAS-dependent cancers. Hence, it is essential that models of KRAS-driven cancers accurately reflect the genetics of human tumors and recapitulate the complex tumor-stromal intercommunication that is manifest in human cancers. See http://gan.sagepub.com/content/2/3/335.full.
It is known in the art that a specific mutation in the KRAS gene, G12D, leads to its constitutive activation. KRAS was identified as an oncogene in 1982 (see Der C. J., et al., Cell. 1983; 32:201-8) and is found to be mutated at a high frequency in human cancers including 95% of pancreatic ductal adenocarcinomas (PDAC), 50% of colon cancers, and 30% of non-small cell lung cancers (NSCLC). Overall, activating mutations in RAS are found in 32% of human cancers, including 21% with KRAS mutation, 8% with N-RAS mutation, and 3% with H-RAS mutation. See, e.g., Bos J. L., Cancer Res. 1989; 49:4682-9; Chang E H, et al., Proc Natl Acad Sci USA. 1982; 79:4848-52.
Mutational activation of KRAS results in aggressive cancers, is generally correlated with poor prognosis in cancers, and is associated with poor response to many existing therapies. See, e.g., Uberall I., et al., Exp Mol Pathol. 2008; 84:79-89; Cappuzzo F., et al., Br J Cancer. 2008; 99:83-9; Eberhard D. A., et al., J Clin Oncol. 2005; 23:5900-9. Despite the early recognition of KRAS as an oncogene, efforts to develop therapies targeting KRAS and KRAS-driven tumors have been largely unsuccessful.
In light of the multitude of effects of KRAS, including intracellular and intercellular interactions, it is critical to understand KRAS-driven tumorigenesis in a setting that recapitulates the complex biology of tumors in patients. While genetically engineered mouse models of cancer have proven to be valuable tools in cancer research, a large animal model that more closely resembles human biology and size would facilitate greater understanding of the processes involved in tumor etiology.
ATM Animal Model Platform
Mutations in the Ataxia-Telangiectasia Mutated (ATM) gene give rise to a condition known as Ataxia-Telangiectasia (A-T), described in co-pending U.S. Appln. No. 61/788,080, filed on Mar. 15, 2013, and PCT/US14/29248, filed on Mar. 14, 2014, each of which is hereby incorporated by references in their entireties. The ATM gene was first identified and cloned in 1995 (see, e.g., Savitsky, K., et al., Science, 1995. 268(5218): p. 1749-53). The ATM gene is 160 kb in length, and encodes a transcript of 13 kb spanning 66 exons. To date, at least 432 unique mutations have been identified in ATM, the majority of which are truncating or splice-site mutations that give rise to shorter, non-functional ATM proteins. ATM is a Ser/Thr protein kinase that is a member of the phosphoinositide 3-kinase (PI3K)-related protein kinase (PIKK) family, as is Rad3-related protein (ATR), both of which are involved in DNA damage response. The kinase domain of ATM is known to act on the tumor suppressor protein p53, both in vitro and in vivo, and activation of p53 is deficient in A-T cells. See, e.g., Banin, S., et al., Science, 1998. 281(5383): p. 1674-7; Canman, C. E., et al., Science, 1998. 281(5383): p. 1677-9; Khanna, K. K., et al., Nat Genet, 1998. 20(4): p. 398-400.
ATM and related proteins are known to play an important role in DNA damage repair, and loss of ATM function results in disruptions in a number of cellular pathways. Cells without any functional ATM protein are hypersensitive to radiation and do not respond normally to DNA damage. Instead of activating DNA repair, the defective ATM protein allows mutations to accumulate in other genes, which may cause cells to grow and divide in an uncontrolled way leading to the formation of cancerous tumors. Additionally, as discussed above, it is known that people who have only one copy of the ATM gene in each cell (ATM +/−) are at an increased risk of developing certain types of cancer.
Murine models of A-T have provided insights into the consequences of ATM dysfunction but do not replicate the full repertoire of clinical symptoms observed in A-T disease or in the progression and development of ATM-related cancers. While these mice are useful for investigating some of the cellular pathways in which ATM is involved, they are not ideal for studying the development of cancers associated with ATM or for testing new therapeutic approaches.
Given the examples provided by p53, ATM and KRAS, among others, a large animal model that shares anatomical, physiological, and developmental similarities with humans and more accurately models cancer development and progression could be a transformative resource, bridging the gap between the current mouse models and the development of effective treatments in humans.
Large Animal Models of Human Cancer
Provided herein are the first gene-targeted large animal models of human cancer, and human cancer development and progression. Analogous mouse models exist and have been extremely useful for understanding cancer biology and early-stage drug development, but an animal that is more similar to humans in size, anatomy, physiology, genetics, and tumor biology would be a uniquely applicable resource. A large animal model would overcome many of the disadvantages inherent in the currently available mice models, particularly with respect to size, lifespan, telomere length, cancer biology, and metabolism. Further, the large animal models disclosed herein are not intended to replace current (and future) murine models, but rather to complement existing efforts in humans and mice and provide an opportunity for multi-species, comparative approaches to fighting and preventing cancer, cancer development and/or progression.
In one example, the large animal model may be generated in, for example, a miniature pig that is more representative of average human size and lifespan (10-15 years). In such an example, porcine tumors will grow at a rate and to sizes observed in people. See, e.g., Adam, S. J., et al., Oncogene 2007, 26 (7), 1038-45.
In addition to the disadvantages of murine cancer models discussed above, another shortcoming of murine models of cancer is that mice have much longer telomeres than humans (40-60 kb vs. 10-15 kb) due to the presence of telomerase activity in adult cells. See, e.g., Rangarajan, A., et al., Nat Rev Cancer 2003, 3 (12), 952-9. In large animals, for example, in pigs, as in humans, there is little post-embryonic cellular telomerase activity, resulting in, e.g., porcine telomeres that are 15-20 kb in length. See, e.g., Jiang, L., et al., Biol Reprod 2004, 70 (6), 1589-93. Furthermore, porcine telomerase undergoes reactivation in cancer cells. See, e.g., Pathak, S.; Multani, A. S., et al., Int J Oncol 2000, 17 (6), 1219-24. Accordingly, large animal models, such as pigs, may provide a more appropriate setting for modeling the transformative events that occur in human tumorigenesis. In another example, a side by side comparison of common cancer-related genes (including TP53 and KRAS) in human, porcine, and murine fibroblasts found that pigs, like humans (but unlike mice), are highly resistant to tumorigenesis and require a similar molecular combination of genetic changes to promote cancer. See Adam, S. J., et al., Oncogene 2007, 26 (7), 1038-45.
Further, the large animal models disclosed herein are also more suitable with regard to metabolism. Mice metabolize drugs differently than humans, for example, in the processing of oxidants and mutagens (Rangarajan, A., et al., Nat Rev Cancer 2003, 3 (12), 952-9). This makes it difficult to accurately assess drug safety and toxicity, and may also explain why so many cancer therapies are successful in mice, but fail in humans. In contrast, a recent survey of 150 compounds revealed that large mammals were more predictive for human toxicity compared to rodents (63% versus 43%). See Olson H, et al., Regulatory toxicology and pharmacology: RTP 2000; 32(1):56-67 (doi: 10.1006/rtph.2000.1399. PubMed PMID: 11029269). Moreover, investigations in large animal models can be performed in a relevant, diseased setting with normal immunological response to the tumor.
One example of this is the cytochrome P450 CYP3A. Nearly half of prescription drugs are metabolized by CYP3A. See Maurel, P., CRC Press: 1996; p 241-270. While, for example, human and porcine CYP3A have similar catalytic selectivity for numerous compounds, rodent CYP3A fails to metabolize a number of common prodrugs. See, e.g., Soucek, P., et al., BMC Pharmacol 2001, 1, 11; Guengerich, F. P., Chem Biol Interact 1997, 106 (3), 161-82. Moreover, gene regulation of CYP3A in response to drugs (or other xenobiotics) greatly dictates how a drug is metabolized. A key xenosensor regulating CYP3A expression is pregnane X receptor (PXR), and porcine PXR is highly similar to its human counterpart. See, e.g., Xie, W., et al., Nature 2000, 406 (6794), 435-9; Moore, L. B., et al., Mol Endocrinol 2002, 16 (5), 977-86. This is not unexpected because, drugs are metabolized by a system that evolved to combat dietary xenobiotics, and humans and pigs are both true omnivores whereas rodents are herbivores. See Xie, W., et al., Drug Discov Today 2002, 7 (9), 509-15. Finally, the basal metabolic rate (BMR) in mammals is typically related to body size, and consequently humans and, e.g., pigs share a similar BMR, whereas the BMR of mice is seven times higher than in humans. See, e.g., Randall, D., Ekert Animal Physiology: Mechanisms and Adaptions, 5th ed.; W.H. Freeman and Company: 2001; Ames, B. N., et al., Proc Natl Acad Sci USA 1993, 90 (17), 7915-22. Accordingly, larger animals models, including a porcine model, may be more appropriate preclinical models for drug toxicity than rodents. In fact, in a recent survey of 150 compounds revealed that large mammals were more predictive for human toxicity compared to rodents (63% versus 43%). See Olson, H., et al., Regul Toxicol Pharmacol 2000, 32 (1), 56-67.
The large animal models disclosed herein allow for testing of therapeutic approaches to cancer that are impossible in smaller animals, such as intensity-modulated radiation and local hyperthermia. A large animal model (for example, a porcine model), will also serve an unmet need in medical imaging and surgical training. For example, noninvasive image-guided technologies including next generation MRI, ultrasound, nuclear imaging, x-ray and optical imaging techniques could be evaluated in, e.g., pigs using instrumentation designed for humans. In the context of surgical training, a large animal model (for example, a porcine model) would allow refinement of surgical techniques using standard approaches, as well as minimally invasive and robotic technologies. Further, these investigations would all be performed in a relevant, diseased setting.